G Model
EURO-8513; No. of Pages 4 European Journal of Obstetrics & Gynecology and Reproductive Biology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
An evidence in vitro for the influence of bisphenol A on uterine leiomyoma Yang Shen a,*, Mu-Lan Ren a, Xu Feng a, Yun-Lang Cai a, Yong-Xing Gao a, Qian Xu b,c a
Department of Obstetrics and Gynecology, Zhongda Hospital, School of Medicine, Southeast University, Nanjing 210009, China School of Public Health, Southeast University, Nanjing 210009, China c Ministry of Education Key Laboratory of Environmental Medicine Engineering, Southeast University, Nanjing 210009, China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 January 2014 Received in revised form 28 March 2014 Accepted 31 March 2014
Objective: To study the effect and mechanism of environmental estrogens bisphenol A (BPA) on uterine leiomyoma (UL) cells. Methods: Primary cultures and subcultures of human UL cells, which were identified by immunocytochemical staining with a monoclonal anti-a-smooth muscle actin antibody, were performed. The viability of cells in different concentration of bisphenol A of 24 h, 48 h and 72 h were analyzed by CCK-8. The expressions of mRNA of ERa, IGF-1 and VEGF in all groups were detected by real-time quantitative PCR assay, and then the expressions of proteins detected by Western blot assay. Results: BPA promoted the growth of UL cells and the expressions of ERa, IGF-1 and VEGF, which had positive correlation with the concentration and action time of BPA treatment. Conclusion: BPA promotes the growth of leiomyoma cells. The expressions of IGF-1, VEGF can be upregulated by ERa, which may be possible mechanism of BPA promote the growth of leiomyoma cells. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Uterine leiomyoma Environmental estrogen Bisphenol A ERa IGF-1 VEGF
Introduction Uterine fibroid is the most common benign tumor in the female reproductive system, and also the most common cause of hysterectomy in premenopausal women [1]. As the incidence of uterine leiomyoma is increasing, attention must be paid to the prevention of uterine leiomyoma. But the causes of uterine leiomyoma remain unknown, most gynecological researchers support the hypothesis that uterine leiomyoma is hormonally responsive tumors [2]. With the increase of environmental estrogens (EEs) pollution, the concentration of bisphenol A (BPA) has become the primary indicator of phenolic environmental estrogens [3]. Recent studies showed that EEs make some influence on the development of uterine fibroids [4], which remain uncertain. In this study, we observed the changes in uterine leiomyoma (UL) cell activity and investigated gene and protein expression changes of ERa, IGF-1, VEGF, by using different concentrations of BPA acting on the UL cells, to explore the role of environment estrogen in promoting the growth of uterine fibroids and its
* Corresponding author. Tel.: +86 15366166769. E-mail address: [email protected] (Y. Shen).
possible mechanism of action. The results are expected to provide theoretical basis for the clinical prevention and treatment of uterine fibroids. Materials and methods Tissue collection Leiomyoma samples were obtained from premenopausal women undergoing hysterectomy at Department of Gynecology and Obstetrics in Zhongda Hospital, Southeast University. Permission to use these samples was approved by the Ethics Committee of Southeast University. All samples were obtained after receiving written informed consent from the patients. Culturing of human UL cells Upon retrieval from the operating room, the specimens of uterine fibroid was diced into several blocks, about 1 cm3 size of each. Care was taken to avoid including any portion of adjacent myometrium, and blocks were immediately placed into D-Hank’s medium in a separated container and immediately removed to the laboratory under low temperature. The cell culture procedure was referenced to reported method [5]. The morphology of cultured
http://dx.doi.org/10.1016/j.ejogrb.2014.03.052 0301-2115/ß 2014 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: Shen Y, et al. An evidence in vitro for the influence of bisphenol A on uterine leiomyoma. Eur J Obstet Gynecol (2014), http://dx.doi.org/10.1016/j.ejogrb.2014.03.052
G Model
EURO-8513; No. of Pages 4 Y. Shen et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology xxx (2014) xxx–xxx
2
cells was carried out in an inverted phase contrast microscope, and the smooth muscle actin a (a-actin) for cells identification was detected by immunocytochemistry method. Cells used in the experiments were passaged twice or three times.
developed by an X-Ray developer. The intensity of each protein band was quantified by using image analysis software and normalized against corresponding b-actin, detected by anti-bactin antibody (1:5000), and then the normalized ratio was used to generate data graphs.
Observation of cell viability Statistical analysis Leiomyoma cells were trypsinized and suspended, from which 2 105 well1 were seeded in 96 well culture plates and allowed to grow for 24 h at 37 8C. Ten microliter BPA with various concentrations was mixed into the cultured wells after 24 h later of adherent cells. According to the literature reported at the concentration range of proliferation effect of BPA [6–8], seven experimental concentrations were determined as 0 mmol/l with medium only, 0 mmol/l with same amount of 1% ethanol, 1 mmol/l, 2.5 mmol/l, 5 mmol/l, 10 mmol/l, 20 mmol/l. Each concentration was set up for five wells. The culture medium was centrifuged and then the supernatant was discarded, respectively, at 24, 48 and 72 h of cell culture. Ten microliter of WST-8 was added into each well, which was keep in incubator for 0.5 to 4 h at 37 8C in a humidified atmosphere of 95% air and 5% CO2. Subsequently, the proliferation rate (PR) was calculated according to the interpretation of value at the 450 nm. Expressions of ERa mRNA, IGF-1 mRNA, VEGF mRNA with real-time quantitative PCR assay Total RNA was prepared from UL Cells and treated with BPA or vehicle medium using an RNA extraction kit (Qiagen, Valencia, CA). One microgram of total RNA from each sample was reversetranscribed and an equal amount of cDNA (40 ng) was used for amplification of ERa, IGF-1 or VEGF (Applied Biosystems) and detected real-time by Bio-Rad IQ5. Real-time PCR reaction system was designed as follows: SYBR Green Mix 10 ml, the upstream primer 0.6 ml, downstream primer 0.6 ml, ddH2O 6.8 ml, cDNA 2 ml, the total volume of 20 ml. Meanwhile, the amplification reaction conditions were set for 35 cycles as follows: predenaturation at 94 8C for 2 min, then denaturation at 94 8C for 45s, annealing at 56 8C for 45 s, extension at 72 8C for 45 s. Finally, the fluorescence data was collected for analysis. The primer sequences used for ERa, IGF-1 and VEGF quantification are as follows: ERa forward, 50 -TGT GCA ATG ACT ATG CTT CA-30 ; ERa reverse, 50 -GCT CTT CCT CCT TGT TTT TA-30 ; IGF-1 forward, 50 -GCT GGT GGA TGC TCT TCA GTT C-30 ; IGF-1 reverse, 50 -AGCTGACTTGGCAGGCTTGAG-30 ; VEGF forward, 50 -GCA GAA TCA TCA CGA AGT GGT-30 ; and VEGF reverse, 50 -TGA AGA TGT ACT CGA TCT CAT CA30 .
Data analysis was performed by using SPSS software (SPSS Inc., Chicago, IL, USA). All of the result data was expressed as standard deviation of the mean (SD). One-way ANOVA and LSD (Leastsignificant difference) methods were used for comparison between groups. Bivariate correlation analysis was processed by Pearson correlation coefficient method. A P-value of
EURO-8513; No. of Pages 4 European Journal of Obstetrics & Gynecology and Reproductive Biology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
An evidence in vitro for the influence of bisphenol A on uterine leiomyoma Yang Shen a,*, Mu-Lan Ren a, Xu Feng a, Yun-Lang Cai a, Yong-Xing Gao a, Qian Xu b,c a
Department of Obstetrics and Gynecology, Zhongda Hospital, School of Medicine, Southeast University, Nanjing 210009, China School of Public Health, Southeast University, Nanjing 210009, China c Ministry of Education Key Laboratory of Environmental Medicine Engineering, Southeast University, Nanjing 210009, China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 January 2014 Received in revised form 28 March 2014 Accepted 31 March 2014
Objective: To study the effect and mechanism of environmental estrogens bisphenol A (BPA) on uterine leiomyoma (UL) cells. Methods: Primary cultures and subcultures of human UL cells, which were identified by immunocytochemical staining with a monoclonal anti-a-smooth muscle actin antibody, were performed. The viability of cells in different concentration of bisphenol A of 24 h, 48 h and 72 h were analyzed by CCK-8. The expressions of mRNA of ERa, IGF-1 and VEGF in all groups were detected by real-time quantitative PCR assay, and then the expressions of proteins detected by Western blot assay. Results: BPA promoted the growth of UL cells and the expressions of ERa, IGF-1 and VEGF, which had positive correlation with the concentration and action time of BPA treatment. Conclusion: BPA promotes the growth of leiomyoma cells. The expressions of IGF-1, VEGF can be upregulated by ERa, which may be possible mechanism of BPA promote the growth of leiomyoma cells. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Uterine leiomyoma Environmental estrogen Bisphenol A ERa IGF-1 VEGF
Introduction Uterine fibroid is the most common benign tumor in the female reproductive system, and also the most common cause of hysterectomy in premenopausal women [1]. As the incidence of uterine leiomyoma is increasing, attention must be paid to the prevention of uterine leiomyoma. But the causes of uterine leiomyoma remain unknown, most gynecological researchers support the hypothesis that uterine leiomyoma is hormonally responsive tumors [2]. With the increase of environmental estrogens (EEs) pollution, the concentration of bisphenol A (BPA) has become the primary indicator of phenolic environmental estrogens [3]. Recent studies showed that EEs make some influence on the development of uterine fibroids [4], which remain uncertain. In this study, we observed the changes in uterine leiomyoma (UL) cell activity and investigated gene and protein expression changes of ERa, IGF-1, VEGF, by using different concentrations of BPA acting on the UL cells, to explore the role of environment estrogen in promoting the growth of uterine fibroids and its
* Corresponding author. Tel.: +86 15366166769. E-mail address: [email protected] (Y. Shen).
possible mechanism of action. The results are expected to provide theoretical basis for the clinical prevention and treatment of uterine fibroids. Materials and methods Tissue collection Leiomyoma samples were obtained from premenopausal women undergoing hysterectomy at Department of Gynecology and Obstetrics in Zhongda Hospital, Southeast University. Permission to use these samples was approved by the Ethics Committee of Southeast University. All samples were obtained after receiving written informed consent from the patients. Culturing of human UL cells Upon retrieval from the operating room, the specimens of uterine fibroid was diced into several blocks, about 1 cm3 size of each. Care was taken to avoid including any portion of adjacent myometrium, and blocks were immediately placed into D-Hank’s medium in a separated container and immediately removed to the laboratory under low temperature. The cell culture procedure was referenced to reported method [5]. The morphology of cultured
http://dx.doi.org/10.1016/j.ejogrb.2014.03.052 0301-2115/ß 2014 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: Shen Y, et al. An evidence in vitro for the influence of bisphenol A on uterine leiomyoma. Eur J Obstet Gynecol (2014), http://dx.doi.org/10.1016/j.ejogrb.2014.03.052
G Model
EURO-8513; No. of Pages 4 Y. Shen et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology xxx (2014) xxx–xxx
2
cells was carried out in an inverted phase contrast microscope, and the smooth muscle actin a (a-actin) for cells identification was detected by immunocytochemistry method. Cells used in the experiments were passaged twice or three times.
developed by an X-Ray developer. The intensity of each protein band was quantified by using image analysis software and normalized against corresponding b-actin, detected by anti-bactin antibody (1:5000), and then the normalized ratio was used to generate data graphs.
Observation of cell viability Statistical analysis Leiomyoma cells were trypsinized and suspended, from which 2 105 well1 were seeded in 96 well culture plates and allowed to grow for 24 h at 37 8C. Ten microliter BPA with various concentrations was mixed into the cultured wells after 24 h later of adherent cells. According to the literature reported at the concentration range of proliferation effect of BPA [6–8], seven experimental concentrations were determined as 0 mmol/l with medium only, 0 mmol/l with same amount of 1% ethanol, 1 mmol/l, 2.5 mmol/l, 5 mmol/l, 10 mmol/l, 20 mmol/l. Each concentration was set up for five wells. The culture medium was centrifuged and then the supernatant was discarded, respectively, at 24, 48 and 72 h of cell culture. Ten microliter of WST-8 was added into each well, which was keep in incubator for 0.5 to 4 h at 37 8C in a humidified atmosphere of 95% air and 5% CO2. Subsequently, the proliferation rate (PR) was calculated according to the interpretation of value at the 450 nm. Expressions of ERa mRNA, IGF-1 mRNA, VEGF mRNA with real-time quantitative PCR assay Total RNA was prepared from UL Cells and treated with BPA or vehicle medium using an RNA extraction kit (Qiagen, Valencia, CA). One microgram of total RNA from each sample was reversetranscribed and an equal amount of cDNA (40 ng) was used for amplification of ERa, IGF-1 or VEGF (Applied Biosystems) and detected real-time by Bio-Rad IQ5. Real-time PCR reaction system was designed as follows: SYBR Green Mix 10 ml, the upstream primer 0.6 ml, downstream primer 0.6 ml, ddH2O 6.8 ml, cDNA 2 ml, the total volume of 20 ml. Meanwhile, the amplification reaction conditions were set for 35 cycles as follows: predenaturation at 94 8C for 2 min, then denaturation at 94 8C for 45s, annealing at 56 8C for 45 s, extension at 72 8C for 45 s. Finally, the fluorescence data was collected for analysis. The primer sequences used for ERa, IGF-1 and VEGF quantification are as follows: ERa forward, 50 -TGT GCA ATG ACT ATG CTT CA-30 ; ERa reverse, 50 -GCT CTT CCT CCT TGT TTT TA-30 ; IGF-1 forward, 50 -GCT GGT GGA TGC TCT TCA GTT C-30 ; IGF-1 reverse, 50 -AGCTGACTTGGCAGGCTTGAG-30 ; VEGF forward, 50 -GCA GAA TCA TCA CGA AGT GGT-30 ; and VEGF reverse, 50 -TGA AGA TGT ACT CGA TCT CAT CA30 .
Data analysis was performed by using SPSS software (SPSS Inc., Chicago, IL, USA). All of the result data was expressed as standard deviation of the mean (SD). One-way ANOVA and LSD (Leastsignificant difference) methods were used for comparison between groups. Bivariate correlation analysis was processed by Pearson correlation coefficient method. A P-value of
